This program is designed to help understand the role played by the pteridine cofactor, tetrahydrobiopterin, in the mixed function oxygenase reactions of phenylalanine and tyrosine hydroxylase. During the course of these reactions one atom of molecular oxygen is inserted into the aromatic ring of phenylalanine or tyrosine with a synchronous conversion of the tetrahydropterin to an "active" or "dihydro quinonoid" form of the pterin. Pterin reductase, in the presence of NADH, converts the dihydropterin back to the tetrahydro level. With the aid of affinity chromatography the enzymes have been isolated in homogeneous form from rat liver. Their main structural features are being investigated with the use of polyacrylamide electrophoresis, gel filtration, specific amino acid blocking agents and circular dichroism techniques. Analogs of the amino acid substrates containing photoaffinity labels are to be synthesized to aid in the characterization of the hydroxylase active sites, and in addition, previously synthesized fluorescent folate analogs are to be used to probe the pterin binding site of the reductase.